|Year : 2015 | Volume
| Issue : 3 | Page : 98-101
Acetylcholinesterase activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia: An in vitro approach
Konstantinos Kalafatakis1, Vasiliki Gkanti2, Connie A Mackenzie-Gray Scott3, Apostolos Zarros2, George S Baillie3, Stylianos Tsakiris4
1 Laboratory of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK
2 Laboratory of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Gardiner Laboratory, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
3 Gardiner Laboratory, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
4 Laboratory of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
|Date of Web Publication||28-Sep-2015|
Dr. Stylianos Tsakiris
Laboratory of Physiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, Goudi, GR - 11527 Athens
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Hyperprolinaemia is characterized by increased tissue accumulation of proline (Pro) and is known to exert serious cognitive and/or neuropsychiatric symptomatology as a direct result of Pro accumulation in the brain. The aim of this study was to explore a putative link between experimentally-simulated hyperprolinaemia and the activity of acetylcholinesterase (AChE); a crucial neurotoxicity marker. In vitro experiments were undertaken on purified eel-derived AChE, as well as on adult mouse brain homogenates, in order to examine the effect of a spectrum of Pro concentrations (3, 30, 500, and 1000 μM) on this marker. Our data showed that although Pro exerted a significant inhibitory effect on pure AChE activity, mouse brain-derived membrane-bound AChE activity was found either unaltered or significantly increased following incubation with Pro. The use of AChE activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia should be considered with caution and in parallel with a number of other experimental parameters.
Keywords: Acetylcholinesterase, hyperprolinaemia, neurotoxicity, proline
|How to cite this article:|
Kalafatakis K, Gkanti V, Mackenzie-Gray Scott CA, Zarros A, Baillie GS, Tsakiris S. Acetylcholinesterase activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia: An in vitro approach. J Nat Sc Biol Med 2015;6, Suppl S1:98-101
|How to cite this URL:|
Kalafatakis K, Gkanti V, Mackenzie-Gray Scott CA, Zarros A, Baillie GS, Tsakiris S. Acetylcholinesterase activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia: An in vitro approach. J Nat Sc Biol Med [serial online] 2015 [cited 2018 Aug 20];6, Suppl S1:98-101. Available from: http://www.jnsbm.org/text.asp?2015/6/3/98/166099
The term "hyperprolinaemia" describes two distinct inherited metabolic disorders resulting in increased tissue accumulation of proline (Pro): Hyperprolinaemia type I and II.  The first occurs due to Pro oxidase deficiency [Figure 1]a, while the second is caused by the functional inadequacy of Δ 1 -pyrroline-5-carboxylic acid dehydrogenase [Figure 1]a; both disorders are associated with elevated Pro levels in the central nervous system (CNS), and in some cases can lead to the manifestation of epileptic, serious cognitive (e.g., mental retardation), and/or other neuropsychiatric symptomatology. , However, the neurochemical mechanisms underlying these symptoms are poorly understood, while, to date, the experimental efforts of reliably simulating the CNS of hyperprolinaemic states have only resulted in certainty of the role of oxidative stress in its etiopathogenesis. 
In recent years, a number of studies have focused on the in vivo and in vitro effects of Pro on rat CNS acetylcholinesterase (AChE) activity. ,, Apart from its classical role in hydrolyzing acetylcholine, AChE is an extensively studied enzyme of paramount importance for synaptic integrity, neurite outgrowth, neurodevelopment, and apoptosis. Delwing et al.  injected 12-day-old Wistar rats with a single subcutaneous injection of Pro (12.8 μmol/g of body weight) and subsequently isolated their cerebral cortices 1 h after the injection; through this approach, the rats were reported to have achieved plasma Pro levels of 1-2 mM (similar to those found in type II hyperprolinaemic patients) and CNS Pro levels of 0.25-0.55 mM. This acute administration of Pro resulted in a significant inhibition of rat cerebral AChE activity (−25% as compared to controls) and was prevented by pretreatment with established antioxidants (vitamin C or E).  A few years later, Delwing et al.  repeated the same experiment on 29-day-old Wistar rats receiving a subcutaneous injection of Pro at a higher concentration (18.2 μmol/g of body weight), and confirmed the significant inhibition of cerebral AChE activity due to Pro (−32% as compared to that of controls). However, the undertaking of a more extended treatment scheme (where Pro was administered subcutaneously twice a day at 10 h intervals from day 6 to day 28 of age, at increasing doses ranging from 12.8 to 18.2 μmol/g of body weight) has failed to exert any significant effect on the rat cerebral AChE activity, despite the fact that the rats achieved (again) plasma Pro levels similar to those of hyperprolinaemic type II patients (1-2 mM).  These findings formed the basis of a more recent study on male Wistar rats by Ferreira et al.  that used the same increasing dose Pro administration scheme but (i) either allowed for a washout period prior to the sacrifice of the rats on 60 th day of age or (ii) continued to administer Pro at the dose of 18.2 μmol/g of body weight from day 29 to day 60 of age (where sacrifice also occurred). Both treatment schemes failed to produce any significant changes with regards to AChE activity in the cerebral cortex of Pro-treated rats (as compared to saline-treated controls), but led to a significant increase of hippocampal AChE activity in hyperprolinaemic rats (as compared to controls). 
The effect of Pro administration on AChE activity has also been the subject of the recent study by Savio et al.  on zebrafish (Danio rerio). Exposure at two Pro concentrations (1.5 and 3 mM for 1 h or 1-week) revealed that zebrafish brain AChE activity can only be significantly altered following the long-term (1-week) exposure to Pro (+34% and +39% after exposure to 1.5 and 3 mM Pro, respectively, as compared to controls).  Interestingly, Savio et al.  also found that these increases in AChE activity were accompanied by significantly depressed expression of the ache gene, as demonstrated by quantitative real-time reverse transcription polymerase chain reaction assays; a finding suggesting that posttranslational events are likely to contribute to the Pro-induced effects on brain AChE activity.
Unfortunately, to date, the undertaking of in vitro experiments has not been of particular use toward the clarification of hyperprolinaemia-induced effects on brain AChE activity. , The incubation of 12-day-old rat cerebral cortices' homogenates at 37°C for 1 h with various concentrations of Pro (3, 30, 500, and 1000 μM) demonstrates a concentration-dependent inhibition of AChE that was significant after exposure to 0.5 and 1 mM Pro and reversible by vitamin E co-exposure.  On the other hand, the in vitro effect of the same Pro concentration spectrum on zebrafish brain homogenates (tested at 25°C) did not affect AChE activity at all. 
We herein present our findings with regards to the in vitro effect of exactly the same spectrum of Pro concentrations on pure eel-derived AChE activity [Figure 1]b as well as on adult female mouse (C57BL/6) brain homogenates' AChE activity following a 30 min, 1 h and 3 h incubation period [Figure 1]c. The determination of AChE activity was performed according to the method of Ellman et al.  as previously described in detail,  while brain samples' protein determination was performed, according to the method of Lowry et al.  Obtained data were analyzed using one-way ANOVA followed by Bonferroni correction (where applicable), performed by SPSS (16.0) for Windows Software. Our data show: (i) that Pro provokes a direct, statistically significant inhibition of AChE activity at all tested concentrations [Figure 1]b and (ii) that adult murine brain homogenates exposed to any Pro concentration after a 3 h incubation present with a significant increase of AChE activity (as compared to controls), in contrast to shorter periods of incubation (30 min and 1 h) where this increase is insignificant [Figure 1]c.
These findings indicate that although Pro exerts a significant (concentration-dependent) inhibitory effect on pure AChE activity, brain-derived membrane-bound AChE activity is either unaltered or significantly increased following an incubation with Pro; an effect that introduces the possibility of Pro-induced modification of the lipid(s) - AChE interactions as a regulator of AChE activity as in the case of other experimentally-simulated encephalopathies at the in vitro level.  Considering the fact that Pro has also been found to provoke lipid peroxidation (as determined by an increase of thiobarbituric acid reactive species levels in the Pro-treated rat brain homogenates) following both acute  and long-term  in vivo exposure of very young rats to the amino acid, the role of oxidative stress as an inhibitor of AChE  proves to potentially be a major contributor to the reported in vivo acute Pro-induced AChE inhibition. , However, oxidative stress is probably irrelevant with regards to the increase in membrane-bound AChE activity following a long-term in vivo exposure to Pro  or an in vitro simulation of hyperprolinaemia as attempted on adult brain tissue by Savio et al.  and ourselves [Figure 1]c.
The current brief report summarizes the findings of an in vitro approach to the study of AChE activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia, and has not reproduced any of the in vivo approaches (either acute or chronic) already reported in the literature. We believe that AChE activity should be considered as an in vivo neurotoxicity marker based on: (i) the extent of the treatment/exposure to Pro, (ii) the age/maturity of the laboratory animals' CNS at the sacrifice time point (as hyperprolinaemia seems to exert an age-dependent neurotoxicity), (iii) the extent of the undergoing lipid peroxidation and metabolic activity in hyperprolinaemic CNS tissues (parameters that must be co-examined in order to clarify the nature of the observed neurochemical changes), as well as (iv) a closer study on the effects of experimentally-induced hyperprolinaemia on sodium-potassium adenosine triphosphatase (Na + , K + -ATPase) activity , and/or other markers of excitotoxicity,  that could well correlate with changes in AChE activity.
Finally, the examination of a wider CNS-region spectrum under both in vivo and in vitro conditions could shed more light on the nature and diversity of the effects of experimentally-simulated hyperprolinaemia on the activity of AChE; such an undertaking could provide us with more reliable tools toward the representation of this entity at an experimental level, which in turn may influence putative therapeutic strategies to counteract hyperprolinaemia.
The authors would like to acknowledge their appreciation to Dr. Christina Elliott (University of Glasgow) for her assistance in obtaining the necessary mouse brain tissues.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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